Objective:
To identify enzymes that augment the digestibility of forages by ruminal microorganisms.

Approach:
Two approaches will be taken to identify novel enzymes for improving ruminal degradation of plant biomass: 1) The first will involve screening of enzymes or enzyme mixtures from non-ruminal sources for their ability to improve the in vitro fermentation of biomass materials by ruminal inocula. 2) The second will involve the identification of combinations of ruminal inocula, obtained from different cows, that display synergistic improvement of fermentability. The individual donor inocula that, upon combination, display the greatest synergy can subsequently serve as subjects for proteomic investigations to identify unique enzymes present in each inoculum that may be responsible for this synergy.
For both approaches, primary screening of biomass fermentation will be conducted by measuring gas production during in vitro fermentation of representative biomass substrates, including stover from corn (a C4 plant), a C3 grass (e.g., timothy), and a legume (e.g., alfalfa). For Approach 1 above, gas production will be compared in the absence or presence of individual candidate enzymes. For Approach 2, gas production from combinations of ruminal inocula will be compared to that obtained from individual inocula; synergy will be defined as the ratio of gas production by the combined ruminal inocula to the average gas production from the individual donor inocula).
For both approaches, the improvement in fermentation by the best candidate enzymes (Approach 1) or ruminal inoculum combinations (Approach 2) will be confirmed by measurement of residual plant cell wall material (as neutral detergent fiber, NDF) and subsequent calculation of NDF digestion (fraction of added NDF removed). For a six-month extension of this project, we will employ an in vitro gas production assay to: 1) screen candidate enzymes for their ability to enhance the fermentation of representative forages by mixed ruminal microorganisms; and 2) screen combinations of ruminal fluids from different donor cows for synergistic improvement of the fermentation of representative forages. In vitro fermentations wth enzymes or ruminal fluids that display enhanced gas production will be further tested for improved conversion of neutral detergent fiber, the primary component of plant cell walls.